The objective of this project is to define events associated with phorbol ester action, beginning with occupancy of the phorbol ester receptor in hormone-responsive cells. Studies have focused on three types of receptor modulation (receptor number, affinity, and coupling to activation of protein kinase C) each of which may contribute to regulation of cellular sensitivity to this class of tumor promoters. First, analysis of receptor properties in cell lysates of control cultures has shown that the receptor is in equilibrium between the cytosol and membrane fractions and that both fractions contribute to the total receptors measured in intact cells. Treatment with phorbol esters shifts the equilibrium to favor the membrane compartment. In cultures pretreated with hormones that cause a decrease in phorbol ester receptor number, there is a decreased ability of phorbol esters to shift this equilibrium. Thus, regulation of receptor number is associated with a decrease in phorbol ester-directed stabilization of the membrane association of the cytoplasmic receptor. Secondly, in intact cells, receptor affinity is temperature-dependent. Studies with the cytoplasmic and membrane receptor in vitro demonstrate the importance of the phospholipid environment of the receptor in regulation of the apparent receptor affinity. Thirdly, certain biological systems show a decrease in responsiveness to phorbol esters in the absence of measurable changes in binding. Investigations of the phospholipid requirements for phorbol ester binding and activation of protein kinase C indicate that in vitro, both activities depend on the lipids used in the assays. Therefore, changes in the phospholipid composition of the membrane may play a role in regulation of the coupling between receptor occupancy and protein kinase C activation.